Recording Neural Activity

RECORDING ION CHANNEL CURRENTS: THE PATCH-CLAMP

Currents through individual ion channels can be recorded using the patch-clamp technique (Fig. 2.10). A fine glass micro-electrode (tip diameter about 1 filled with electrolyte solution is attached to the cell membrane by suction, forming a 'tight seal' (resistance 1 GQ or more, i.e. 109 Q), so that all current flowing through the channel enters the electrode. These currents are very small (a few picoamps, pA) so have to be amplified. The amplifier also incorporates a device for applying a potential to the pipette, so that the potential across the cell membrane at the tip of the pipette can be varied.

Figure 2.10(a) illustrates currents generated by K+ ions flowing through an M-type K+ channel in a ganglion cell membrane. By convention, the direction of current flow always refers to the direction in which +ve ions move. Thus, outward current is generated by +ve ions flowing out of the cell into the pipette (or — ve ions going the other way). Also by convention, outward current is depicted as an upward deflection in the recording. Note that the channel normally adopts one of two states — it is either

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open or shut — but switches spontaneously between the two states. When it is shut, no current flows. When it is open, the current is fairly constant at any given potential. However, when the potential is changed, the amplitude of the channel current changes: this is because the current is given by Ohm's Law:

V = IR so I = V/R, whence the single-channel current i = g(V — Ek)

where g is the single-channel conductance (reciprocal resistance, units = Siemens, S), Vis the membrane potential across the membrane patch and Ek is the equilibrium potential for k+ ions. The conductance is normally constant, and is characteristic for the channel. Single-channel conductances are mostly within the range 2-100 picosiemens (pS): in this case, the conductance is about 8pS with 2.5 mM [k+] in the pipette solution.

This channel is voltage-sensitive — that is, its activity is increased when the membrane is depolarised. Thus, the channel opens very infrequently and for very short periods at —50 mV, but opens more frequently and for longer times at — 30 mV. This activity is expressed by the open probability (Po), that is, the probability that, at any given time, the channel is open (or, in other words, the proportion of time the channel spends in the open state). In the example illustrated, Po was 0.02 at — 50 mV and 0.27 at — 30 mV.

FROM SINGLE-CHANNEL CURRENTS TO WHOLE-CELL CURRENTS

Figure 2.10(a) shows the activity of a single channel. There are many hundreds or thousands of such channels in the entire membrane of a single ganglion cell. The currents through all of these channels add up to give the whole-cell current. This can be recorded using the patch pipette by filling the pipette with a solution of similar ionic composition to that of the cytoplasm (i.e. with high [k+] and low [Na+] and [Ca+]), then rupturing the membrane under the pipette tip (with pressure) once a seal has been established, or by adding an antibiotic ionophore such as nystatin or amphotericin to the pipette solution and letting these diffuse into the membrane under the pipette tip. In the former case, the solution in the pipette is in direct contact with the cytoplasm, so substances in the cytoplasm diffuse into the pipette and vice versa; nystatin and amphotericin conduct small ions such as Na+ and k+ across the cell membrane under the pipette tip, so providing good electrical contact with the cytoplasm, but do not permit total mixing of the two solutions. An older, but still useful, method is to insert one or more fine micro-electrodes filled with a strong k+ solution into the cell and then let them seal into the membrane.

Figure 2.8 (opposite) Effects of the T-type Ca2+ current on the firing behaviour of guinea-pig thalamic relay neurons. (a) Dependence of firing behaviour on membrane potential. At a hyperpolarised potential (—75 mV), a current injection produces a brief burst of action potentials superimposed whereas at — 53 mV the cell responds with a sustained train of action potentials. (Adapted from Fig. 2 in Jahnsen, H and Llinas, R (1984) J. Physiol. 349: 205-226. Published for the Physiological Society by Cambridge University Press.) (b) Interpretation of the records in (a). Each record show voltage-trace (top), injected current pulse (middle) and T-type Ca2+ current (bottom). At the hyperpolarised potential (record a), the T-type Ca2+ current is de-inactivated ('primed'), so a depolarising current pulse opens T-channels to produce a 'Ca2+ spike' with superimposed Na+ spikes. The Na+ spike can be blocked with tetrodotoxin (TTX: record c), leaving a 'pure' Ca2+ spike and T-current. The T-current is transient and inactivates, so terminating the burst. At a depolarised potential (b and d), the T-channels are fully inactivated so depolarisation does not initiate a T-current (record d) and now evokes a train of Na+ spikes instead of a burst (record b). (Computer simulation, adapted from Fig. 24 in Electrophysiology of the Neuron by Hugenaard and McCormick (1994). Published by Oxford University Press, New York — see Further Study)

Figure 2.9 Hyperpolarisation-activated cation current Ih and its role in pacemaking in a guinea-pig thalamic relay neuron. (Adapted from Figs 2 and 14 in McCormick, DA and Pape, H-C (1990) J. Physiol. 431: 291-318. Reproduced by permission of the Physiological Society.) (a) Records showing the time-dependent activation of the h-current by hyperpolarisation and its deactivation on repolarising. (b) Interpretation of rhythmic activity in a thalamic relay neuron. (1) The inter-spike hyperpolarisation activates Ih to produce a slowly rising 'pacemaker' depolarisation. (2) This opens T-type Ca2+ channels to give a more rapid depolarisation, leading to (3) a burst of Na+ spikes (see Fig. 2.8). At (4) the depolarisation has closed (deactivated) the h-channels and has inactivated the T-channels. The membrane now hyperpolarises, assisted by outward K+ current (5). This hyperpolarisation now removes T-channel in-activation and activates Ih (6), to produce another pacemaker potential

Figure 2.9 Hyperpolarisation-activated cation current Ih and its role in pacemaking in a guinea-pig thalamic relay neuron. (Adapted from Figs 2 and 14 in McCormick, DA and Pape, H-C (1990) J. Physiol. 431: 291-318. Reproduced by permission of the Physiological Society.) (a) Records showing the time-dependent activation of the h-current by hyperpolarisation and its deactivation on repolarising. (b) Interpretation of rhythmic activity in a thalamic relay neuron. (1) The inter-spike hyperpolarisation activates Ih to produce a slowly rising 'pacemaker' depolarisation. (2) This opens T-type Ca2+ channels to give a more rapid depolarisation, leading to (3) a burst of Na+ spikes (see Fig. 2.8). At (4) the depolarisation has closed (deactivated) the h-channels and has inactivated the T-channels. The membrane now hyperpolarises, assisted by outward K+ current (5). This hyperpolarisation now removes T-channel in-activation and activates Ih (6), to produce another pacemaker potential

Figure 2.10 M-type K+ channels: from single-channel currents to whole-cell currents, (a) Singlechannel currents recorded from a dissociated rat sympathetic neuron using a cell-attached patch pipette held at estimated membrane potentials of —30 and — 50 mV, (Adapted from Fig, 3 in Selyanko, AA and Brown, DA (1999) Biophys. J. 77: 701-713, Reproduced with permission of the Biophysical Society.) (b) a: a cluster of single-channel openings recorded from a dissociated ganglion cell with a cell-attached patch pipette on stepping for 1 s from an estimated membrane potential of — 50 mV to — 30 mV. b: an averaged 'ensemble' current obtained on averaging the currents generated by 45 steps like that shown in a. c: mean whole-cell current recorded with a nystatin-perforated patch pipette during four steps from —50 to — 30 mV. (Adapted from Fig. 3 in Selyanko, AA et al. (1992) Proc. Roy. Soc. Lond. Ser. B 250: 119-125. Reproduced by permission of The Royal Society)

Figure 2.10 M-type K+ channels: from single-channel currents to whole-cell currents, (a) Singlechannel currents recorded from a dissociated rat sympathetic neuron using a cell-attached patch pipette held at estimated membrane potentials of —30 and — 50 mV, (Adapted from Fig, 3 in Selyanko, AA and Brown, DA (1999) Biophys. J. 77: 701-713, Reproduced with permission of the Biophysical Society.) (b) a: a cluster of single-channel openings recorded from a dissociated ganglion cell with a cell-attached patch pipette on stepping for 1 s from an estimated membrane potential of — 50 mV to — 30 mV. b: an averaged 'ensemble' current obtained on averaging the currents generated by 45 steps like that shown in a. c: mean whole-cell current recorded with a nystatin-perforated patch pipette during four steps from —50 to — 30 mV. (Adapted from Fig. 3 in Selyanko, AA et al. (1992) Proc. Roy. Soc. Lond. Ser. B 250: 119-125. Reproduced by permission of The Royal Society)

Figure 2.10(b) shows the relation between the activity of a small cluster of perhaps five individual M-channels recorded from a small patch of membrane with a cell-attached patch pipette (records a and b) and the whole-cell M-current recorded when the membrane patch under the electrode is permeabilised with nystatin B (record c), as seen when the membrane patch or the whole-cell membrane potential is suddenly stepped from —50 to — 30 mV and back again. As predicted from Fig. 2.10(a), this depolarisation greatly increases the activity of the channels. However, they do not open instantly but instead take many milliseconds to open — that is, their voltage-gating is relatively slow compared to that of (say) a Na+ channel. The time taken by any individual channel to assume its new level of open probability varies stochastically about a mean. This mean value is given by the time constant x (= 1/(1 — e)). This can be estimated for a single channel, or for the small cluster of channels seen in Fig. 2.10(b), by repeating the depolarising step many times, then averaging the currents to give an ensemble current (record b in Fig. 2.10(b)). In this example, the average time-constant after 45 steps was 86ms. The whole-cell current (record c) gives the current through all the channels in the cell membrane, so, since there are several hundred of them, the current is much larger (note that the current scale is 100 times larger) and one now sees an 'averaged' time-course after a single step (though in this experiment four steps were applied and averaged, to obtain a smoother trace). As one might expect, the time-course of the whole-cell current is quite similar to that of the ensemble of the currents through the small cluster of channels. (They may not be exactly the same, since individual channels in different parts of the cell may vary somewhat in behaviour, depending on their local environment.)

FROM CURRENT TO VOLTAGE

Currents through single channels and across the whole cell membrane are recorded under voltage-clamp — that is, the membrane potential is fixed. In a normal cell, however, the voltage is not fixed: the effect of the current is to change the voltage, and signals are normally seen as voltage signals. Figure 2.11 shows how the current through M-channels affects the membrane voltage. When the cell (a frog ganglion cell) was artificially hyperpolarised to — 90 mV (left column) so that all of the M-channels were shut, very little current flowed when the voltage was changed (i.e. the membrane conductance was very low or its resistance was very high) (Fig. 2.11(a)). As a result, when a current was injected across the membrane (Fig. 2.11(b)), there was a large voltage change. (The time-course of this voltage change is dependent on the product of the membrane resistance and capacitance. Membrane capacitance is determined by the lipid composition of the membrane and is relatively constant at around 1 ^/cm2 membrane.) However, when the cell was left to depolarise to its 'natural' level of (in this case) —■46mV (right-hand column), many M-channels were now open. A hyper-polarising step closes some of the channels, giving a slow decline in current, whereas depolarisation opened more, giving a slow increase in current — the gating of M-channels being characteristically slow, as shown in Fig. 2.10. So now when depolarising current is injected into the cell (bottom record), the membrane begins to depolarise as before but the depolarisation opens more M-channels, and the K+ current through these extra M-channels hyperpolarises the membrane nearly back to where it started. Conversely, if one tries to hyperpolarise the membrane by injecting hyperpolarising current, the outward flux of K+ ions diminishes as M-channels close, so the membrane

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